CA2079685C - Synthetic membrane vesicles containing functionally active fusion peptides as drug delivery systems - Google Patents

Synthetic membrane vesicles containing functionally active fusion peptides as drug delivery systems Download PDF

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CA2079685C
CA2079685C CA002079685A CA2079685A CA2079685C CA 2079685 C CA2079685 C CA 2079685C CA 002079685 A CA002079685 A CA 002079685A CA 2079685 A CA2079685 A CA 2079685A CA 2079685 C CA2079685 C CA 2079685C
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vesicles
vesicle
viral
hemagglutinin
drug
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Reinhard Glueck
Peter Klein
Peter Hermann
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Nika Health Products Ltd
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
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Abstract

The phospholipid bi-layer vesicle contains at least one pharmaceutically active drug and comprises cell-specific markers on the membrane which have at least 90% biological activity when measured according to Luescher & Glueck, Antiviral Research 14, 39-50. In the membrane, the cholesterol content is preferably less than 2% by weight, the detergent content preferably less than 1 ppb. The vesicle diameter preferably is about 80 nm. The phospholipid in the membrane may comprise 70 to 95% by weight of phosphatidylcholine and preferably 10 to 20% by weight of phosphatidylethanolamine: preferably 6 to 8% by weight of a crosslinker, preferably of a sulfosuccinimidyl derivate, and at least one cell-specific fusion peptide are linked to the membrane. The vesicles are used for the preparation of pharmaceuticals against AIDS and carcinomas.

Description

Synthetic Membrane Vesicles Containincr Functionally Active Fusion Peptides As Drucr Delivery Systems The invention relates to synthetic membrane vesicles (liposo-mes) as described in the preamble of claim 1, and particular-ly to vesicles exhibiting on their suzvface fusion peptide mo-lecules and other cell-specific proteins. The fusion peptide molecule may be in the form of a synthetic or purified 70 peptide or as a part of a spike glycoprotein molecule of an enveloped virus, e.g: hemagglutinin, such as of influenza, parainfluenza or Semliki Forest virus. The cell-specific protein may be an IgG antibody, e.g. t:he CD4 antibody.
When a drug is given to a subject it must usually pass from the site of administration into the plasma compartment, and therefore the route of administration may have an important effect on the pharmacokinetic profile of the drug in the cir-culation. Thus, oral administration of-_ the drug, while conve-nient, results in a slow onset of drug action and is somewhat unreliable in terms of-achieving optimum plasma drug levels.
By contrast, intravenous injection results in the prompt and exact establishment of circulating levels, at the cost of some pain and inconvenience to the pai:ient. However, even if the drug is injected directly into they systemic circulation, the relationship between administered dose, drug levels and duration of the target site is by no means simple. These parameters are determined by a complex and often competing network of pathways leading either to accumulation of active drug molecules at the target site, or to the inactivation and excretion of the drug. These pathways involve biotransforma-t:ion in the liver and in other tissues, excretion via the kidney or the bile, binding of drugs to fixed or circulating cells or macromolecules, and the passive or mediated passage of the drug across membrane barriers (Greasy, W.A. (1979):

Drug Disposition in Humans, Oxford Univeristy Press, New York).
In recent years, there has been a good deal of interest in the prospect of influencing the distribution and metabolism of drugs in beneficial ways by using various sorts of carrier or drug delivery systems. These systems are designed to con-trol one or more of the following parameters (Juliano, R.R.
(1975), Can. J. Physiol. Pharmaco 56, 683-690):
a) the rate of input of the drug into a particular body compartment;
b) the distribution and localization of the drug in the body;
c) the persistence or rate of metabolism of the drug.
A major improvement in controlled drug delivery systems was the development of liposomes which were first described by Bangkam et al. (Bangkam, A.D., Standi=;h, M.M. and Watkins J.C. (1965), J. Mol. Biol. 13,238-252). Today, the literature claims an extravagant variety of benefits to be gained by de-livering particular drugs in liposomes. These can be loosely grouped under the following headings: .
1. Liposomes may cross biomembranes and may facilitate the transport of drugs through normally impermeable barriers. In particular, liposomes facilitate the intracellular pene-tration of encapsulated compounds.
2. Liposomes may be designed to interact with specific tis-sues, improving drug selectivity and reducing toxicity.
3. Drug pharmacokinetics may be beneficially modified by liposomes, through modulation of drug release, distribution and removal from the systemic circulation..
4. Chemically and metabolically labile drugs may be protec-ted by liposomes from deactivation.

2~~~~~
Drugs of potential therapeutic interest: may be sequestered in this way, the encapsulated compounds exhibiting modified pro-perties, at least in vitro, when compared with the unmodified substances. Unfortunately, early hopes of a revolutionary new approach to chemotherapy have not been completely realised by the experimental facts (Fildes, F.J.T. (1981), Liposomes.
From physical structure to therapeutic applications. Knight (ed.), Elsevier/North-Holland Biomedical Press).
A better result using liposomes as vectors could be achieved by the targetting of liposomes with specific proteins. If substances encapsulated in liposomes were to be delivered more successfully to selected organs o:r tissues, these tar-getting techniques had to be devised i:n order to bypass the accumulation of liposomes at undesirable sites (thus reducing toxicity) and to optimize the delivery to specific cells (thus enhancing the desired effect).
Several investigations have utilized the coating of liposomes with aggregated immunoglobulins in order to optimize delivery to phagocytic cells (Ismail, G., Boxer, L.A. and Bachner, R.L. (1979), Pediatr. Res., 13. 769-773/Finkelstein, M.C., Kuhn, S.H., Schieren, H., Weissmann, G. and Haffstein, S.
(1980): Liposomes and Immunobiology (Tom, B.H. and Six, H.R.
eds.) 255-270. Elsevier/North-Holland Publishing Co., Amster-dam).
A further improvement was described by Gregoriadis and Neerunjun (1975), Biochem. Biophys. Rea. Commun., 65, 537 -544, whereby the targetting of liposomes was enhanced by associating cell-specific IgG antibodies. The uptake of liposomes was augmented 3- to 25-fold when different cell strains were presented with liposomes associated with IgG
immunoglobulins raised against the particular cell strain.
However, the technique employed showed that the liposomes targetted in this way failed to fuse with the cell membrane ,v and therefore an efficient delivery of drugs into the cells was prevented. (Leserman, L., Weinstein, J.N., Blumenthal, R., Sharrow, S.O. and Texy, W.D. (1979), J. Immunol. 122, 585 - 591).
A major improvement in the production of drug delivery sy- -stems was the targetting of liposomes with viral proteins:
liposomal membranes have been reconstituted with proteins such as the hemagglutinin from influenza virus (Almeida, J.D., Brand, C.M., Edwards, D.C. and Heath, T.D. (1975), Lan-cet 2, 899 -901). The efficiency and specificity of early vi-ral interaction with host cells (adsorption, penetration) may be conferred upon liposomal carriers by incorporating the ap-propriate viral proteins into the liposomal membranes. In-deed, the incorporation of Sendai viru:a spike proteins into liposomal biiayers produced at least a 100-fold enhancement in the uptake by mouse L cells of diph~teria toxin fragment A
as compared with fragment A-containing liposomes without spikes (Uehida, T., Kim, J., Yamaizumi, M., Miyahe, Y. and Okada, Y. (1979). Cell. Biol. 80, 10 - 20).
The main drawbacks of the above methods lie in the lack of fully active influenza hemagglutinin fusion peptides. Influ-enza A viruses penetrate their host cells by membrane fusion.
After binding to the cell surface, virus particles are inter-nalized and transported to endosomes and lysosomes. The aci-dic environment in these organelles activates fusion between the viral and host: cell membranes. The advantage of vesicle-cell fusion at low pH lies in the fact that the content of the vesicles is released after internalizing into the cyto-plasma of the cells (pH in the endosome is around 5.2). The discovery of this low-pH-induced fusion of. influenza virus glycoproteins led to many attempts to develop effective drug delivery systems with influenza hemagg~lutinin targetted liposomes.

Kawasaki, K. et al. (Biochemica et Biophysica Acta, (1983), 733, 286 - 290, used reconstituted vesicles of influenza he-magglutinin glycoproteins in egg yolk phophatidylcholine /
spin-labeled phosphatidylcholine / cholesterol (molar ratio 1.6 . 0.4 . 1). Preparations at appropriate protein to lipid ratios (1 . 44 and 1 . 105 mol/mol) contained vesicles with a diameter of 100 - 300 nm and a high densitiy of spikes on the surface, but have several drawbacks:
1) Due to the high cholesterol content and a residue of detergent they show reduced endocytosis by phagocytic cells.
Kawasaki et al themselves report only a 66% activity; but according to a more recently developed, improved test method, the activity is only 10 (!) after 7 minutes (see Example 10 below). This might be due to the fact that Triton X-100 used by Kawasaki et al partially reacts with hemagglutinin and is not completely removed by dialysis.
2) To study the role of the viral membrane components in the fusion reaction in detail, it is necessary t~o be able to manipulate these components. For this purpose a method is required for the isolation and reconstitution of the viral spike proteins, producing reconstituted~virosomes with full biological fusion activity. Despite the efforts of Kawasaki et al., reconstitution of influenza virus envelopes, displaying full biological fusion activity, has not been reported.
3) The attachment of the targetted liposomes containing pharmaceutical active drugs to specific cells could not be demonstrated.
Most of the other methods that have been employed to recon-stitute viral envelopes are based on solubilization of the viral membrane with a detergent and, after sedimentation of the internal viral proteins and genetic material, removal of the detergent from the supernatant. Detergents with a high critical micelle concentration, such as octylglucoside, may be removed effectively by dialysis. Reconstitution employing octylglucoside has been reported for Semliki Forest virus (SFV) {Helenius, A:, Sarvas, M. and Simons, K. (1981), Eur.
J. Biochem. 116, 27 - 35); vesicular stomatitis virus (VSV) (Eidelman, 0., Schlegel, R., Tralka, T.S. and Blumenthal, R.
(1984), J. Biol. Chem. 259, 4622 - 4628); influenza virus (Huang, R.T.C., Wahu, K,., Klenk, H.D. a.nd Rou, R. (1980) Vi-rology, 104, 294 - 302); and Sendai virus (Harmsen, M.D., Wilschut, J., Scherphof, G., Hulstaert, C. and Hoekstra, D.
(1985), Eur. J. Biochem., 149, 591 - 599). However, properly reconstituted viral envelopes were not produced in all cases.
For example, virosomes formed from SFV had a protein to lipid ratio deviating from that of the viral membrane, and viroso-mes produced from VSV did not exhibit biological fusion acti-vity.
Another method, described in the literature, is the detach-ment of influenza viral spikes from the: virus- particles with bromelain. The hemaggiutinin recovered in such a way was at-tached to liposomes (Doms, R., Heleniu;a, A. and White, J.
(1985), J. biol. Chem., 260 (5) 2973 - 2981). The main draw-back of this method is the limited bio7Logical activity of such vesicles due to the cleavage by bz:omelain.
Thus, the technical problem underlying the present invention is to provide vesicles which transport desired drugs or phar-maceutically active substances to specific cells; such as macrophages, T4-helpercells, brain cel:Ls or other cells of specific organs, which then are fully attached to these cells, internalized by phagocytosis or endocytosis so as to -immediately after endocytosis. - delive:r the desired drug into the cytoplasma of the desired cell.
- 7 _ The solution to the above technical problem is achieved by providing a phospholipid bi-layer vesicle having at least one desired drug or pharmaceutically.active substance therein, the vesicle comprising (a) a membrane having one or more viral phosphol:ipids in combination with other phospholipids comprising phosphatidylethanolamine (PE), (b) at least one active fusion peptide that is a non-Sendai viral hemagglutinin that causes the vesicles to be internalized by target cells through phagocytosis or endocytosis, the hemagglutinin being attached directly or indirectly to the membrane, (c) a bifunctional crosslinker linked to phosphatidylet:hanolamine (PE) of the membrane, and (d) at least one cell-specific marker linked via its sulphur to the PE-bound crosslinker, the marker being a biologically active protein for binding to a receptor of target cells.
Preferably, the phospholipid bi-layer vesicle comprises a membrane having at least one functionally active viral fusion peptide on the membrane, optionally together with a viral phospholipid or a residual amount of a non-ionic detergent, or both, and containing a pharmaceutically active substance, wherein (a) the vesicle membrane also comprises a non-viral phospholipid comprising phosphatidyl-ethanolamine; at least one functionally active hemagglutinin selected .from the group consisting of hemagglutinin trimer, hemagglutinin monomer, glycopeptide HA1 and glycopeptide HA2, as a fusion peptide; a - 7a -bifunctional crosslinking agent; and at least one cell-specific marker bound to the membrane; and optionally (b) the vesicle membrane contains cholesterol at a concentration of less than 10% by weight, or a residual amount of the detergent octaethyleneglycol monododecylet.her (OEG) at a concentration of less than to by weight, or both.
Preferably, the vesicle membrane further comprises phosphatidylcholine. In one embodiment, the at least one cell-specific marker, and optionally the fusion peptide is bound to the membrane by the bifunctional crosslinking agent.
It is preferred that the content of cholesterol is less than to by weight and the content of detergent is less than 0.250 by weight. Conveniently, the vesicle diameter is smaller than 100 nm, preferably about 80 nm. The hemagglutinin may stem from influenza virus, rhabdovirus, parainfluenza virus or togavirus, as may the viral phospholipid. Preferab:Ly, the viral phospholipid is present in combination with a 2 to 100-fold quantity of phosphatidylcholine, more preferably a 5 to 15-fold quantity.
Preferably, the phospholipid in the membrane is 70 to 95~
by weight phosphatidylcholine and 5 to 30o by weight, more preferably 10 to 20o by weight, phosphatidylethanolamine, - 7b -and 5 to 10% by weight, more preferably 6 to 8s by weight, relative to the total membrane of a crosslinker.
Preferably, the phospholipid bi-layer vesicle contains at least one substance selected from the group consisting of imidazol-carboxamide, hydroxy-urea, adriblastin, endoxan, fluoro-uracil, colchicine, dextran sulfate, ribonuclease dimes and lysozyme dimes.
The present invention also provides a process for the preparation of a phospholipid bi-layer vesicle comprising at least one fusion peptide and at least one cell-specific marker on the membrane, and at least .one desired drug or pharmaceutically active substance, the process comprising the steps of (a) dissolving purified virus, or parts thereof, containing non--Sendai hemagglutinin that causes the vesicles to be internalized by target cells by phagocytosis or endocytosis, in a non-ionic detergent solution that does not react with hemagglutinin and that comprises octaethyleneglycol monododecylether, (b) subjecting the solution resulting from step (a) to ultracentrifugation, anc~ mixing the resulting supernatant which contains viral lipids and at least one hemagglutinin fusion peptide with the desired drug or substance, (c) combining the mixture with other phospholipids, the other phospholipids comprising phosphatidylethanolamine, (d) repeatedly treating the mixture from step (c) with microcarriers to remove the detergent whereby vesicles are formed, (e) subjecting the vesicles resulting from step (d) to repeated ultrasonicat:ion to adjust the size of the vesicles, (f) reacting t:he vesicles of step (c) with a bifunctional crosslinker for binding to phosphatidyl-ethanolamine (PE) of the vesicle membrane and for binding polypeptides, and pelleting the vesicles, and (g) reacting the pelleted vesicles with a solution containing at least one cell-specific marker for binding to the PE-bound crosslinker, the marker being a biologically active protein for binding to a receptor of target cells.
Preferably, the detergents solution is octaethyleneglycol.
monododeclyether (OEG) and has a concentration of 10 to 250 umol per ml, more preferably 80 to 120 umol per ml.
Conveniently, the detergent is removed from the solution by a three- to four-fold application of 1 to 2g, preferably 1.5g, of polystyrene beaded microcarriers having a mesh size (wet) of 20 to 50 per 100 mg detergent.
Accordingly, viral hemagglutinin virosomes are provided which contain a liposome ideal for endocytosis and a biologically fully active cell-specific marker, preferably a viral hemagglutinin glycoprotein or a derivative thereof, or a synthetic fusion peptide being capable of inducing the immediate fusion of the virosomes after endocytosis by t:he desired cells.

- 7d -In another embodiment of the invention, a suitable crosslinker which adsorbs to the specific liposome is used in the mixture together with a specific antibody, directed to the responsible receptor of the desired cell for inducing the endocytosis mechanism, which is bound to the crosslinker in such a manner that it is still fully biologically active.
The essential feature of these drug delivery vesicles is that they carry on their surface the fully active viral glycoproteins or a derivative thereof and biologically active, specific antibodies being capable of attaching t:o the desired cells, of being internalized by phagocytosis or endocytosis by these cells, inducing the immediate fusion of the vesicles with internal cytoplasmic membranes and releasing the virosome's content into the cytoplasma of these cells. Due to the fully active fusion peptides of the present invention, the drugs are released immediately after phagocytosis so as to avoid an undesired long stay in the endocytosomes which would give rise to unspecific degradation of the pharmaceutical substances contained in the viral hemagglutinin vesicles of the present invention.
At pH 5, the influenza - g _ fusion peptides on the surface of the vesicles are activated in the same way as is the case with live influenza virus. The content of the vesicles is released into the cytoplasma, as is the case with influenza virus and the released nucleo-protein.
The term "liposome" refers to medium sized bilamellar phos-pholipids prepared by controlled detergent removal. The size of the vesicle initially formed upon detergent removal de-10_ pends on the detergent and phospholipid used and, in some cases, on the method and rate of detergent removal.
The present invention also relates to a method of preparing vesicles which are specially suited for phagocytosis. It com-prises the following steps:
1) Dissolution of one or two phospholipids in a non-ionic detergent;
2) vesicle formation through detergent removal with polystyrene beaded micro-carriers (me~~h size - wet - 20-50;
3) a defined mechanical movement is performed during deter-gent removal;
4) the desired diameter of vesicles (50 - 100 nm) is achie-ved by ultrasonification.
In still another embodiment, the present invention refers to vesicles where the phospholipid comprises 70-95%
phosphatidylcholine and 5-30% by weight of another phospholipid, such as phosphatidylethanolamine. The cho-lesterol content is preferably less than 10.
The term "fusion peptide" refers to viral spike glycoproteins containing the fusion peptide. In one embodiment, the present invention refers to the complete hema<~glutinin trimer of vi-ral surface spikes or to one monomer or to one or both clea-ved subunits, the glycopeptides HA1 and HA2, containing the _ g -functional fusion peptide. In another embodiment, the present invention refers to the fusion peptide itself, isolated or synthetically produced. In a particularly preferred embodi-ment of the present invention, these polypeptides, containing the fusion peptide, refer to influenza hemagglutinins, espe-cially the one of the A-HINI subtype.
The term "crosslinker" refers to an organic heterofunctional molecule capable of linking to the surface of vesicles prepa-red according to this invention and capable of binding poly-peptides. In a pref erred embodiment of the present invention, this molecule is an organic, bifunctional molecule containing a carboxylic group and a thiol group, particularly a sulfo-succinimidyl-(S-)derivate, such as S-4~-(p-maleimido-phenyl)-butyrate, S-acetate, S-2-(m-azido-o-ni.trobenzamido)ethyl-1,3' -dithiopropionate, S-6-(4'-azido-2'-ni.trophenylamino)hexan-oate S-(4-azidophenyldithio)propionate, S-2(p-azidosalicyl-amido)ethyl-1,3'-dithiopropionate, S-~'.-(biotinamido)-ethyl-1,3'-dithiopropionate, S-6-(biotinamic~o) hexanoate, S-3-(4-hydroxyphenyl)propionate, S-(4-iodoacetyl)aminobenzoate, S-4-(N-maleimidomethyl)cyciohexane-1-carboxylate, S-2,2,5,5-tetramethylpyrroline-1-oxyl-HC1.
The term "cell-specific" protein or marker referswto a pro-tein capable of linking to the crossiinker at the vesicle's surface and linking to the receptor oj: cells inducing the en-docytosis mechanism. In a preferred ernbodiment of the present invention, this molecule refers to a cell receptor specific antibody, particularly to.a monoclona:L antibody.
The examples and figures illustrate tile invention:
Example 1:
Preparation of synthetic membrane vesicles of phosphatidyl-choline with fully functionally active viral fusion peptides in a hemagglutinin trimer from. influenza virus and containing dextran sulfate as antiviral drug.
Figr-1 shows the principle of the procedure; a circle designates a liquid solution or suspension; a square designates a solid pellet or precipitate. In general:
A fusion buffer solution FB containing 2700 mg solids and a hemagglutinin suspension HA containing 46 mg HA and 31.1 ~unol viral phospholipids VPL were mixed and subjected to ultracen-trifugation UC1. The resulting pellet was dissolved in solut-ion III containing fusion buffer FB and octaethylene glycol monododecyl ether OEG as a mild detergent. Another ultracen-trifugation UC2 yielded a supernatant IV containing FB, HA, VPL and OEG, to which was added the antiviral drug dextran sulfate DS to make up a solution V. In the meantime, a phos-phatidylcholine (PC) solution VI was vacuum dried in a rota-ting round-bottom flask producing a thin PC layer on the in-ner surface of the flask. Solution V was added to this layer and dissolved it to a solution VII which was treated three times with polystyrene beaded microcarriers PBM to remove the OEG and to result in a solution VIII, in which - by ultraso-nification US - vesicles VES of the desired size were developped; the resulting suspension IX was diluted with NaCl solution X to give the drug suspension DR.
In detail:
A fusion buffer solution yI) was prepared containing 7.9 mg 30, NaCl/ml, 4.4 mg/ml trisodiumcitrat dehydrate, 2.1 mg/ml 2-morpholinoethane sulfonic acid monohydrate {MES), and 1.2 mg/ml N-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid in H20 (pH adjusted with 1-N NaOH to 7.3).
Influenza virus-{strain A/ Shanghai / 16/ 89 (H3N2)) was grown in the allantoic cavity of hen eggs, and was purified twice by ultracentrifugation in a sucrose gradient, as de-scribed by Skehel and Schild 1971 (Virology 44, 396 - 408).
The purified influenza virion suspension (II) contained 266 ug of influenza virus hemagglutinin pez- ml and a total of 31.1 p.mol of viral phospholipids, which were determined as follows:
Viral phosphoiipids were extracted according to Folch et al.
(Folch, J., Lees, M. and Sloane, S.G.H.. (1957); J. Biol.
Chem. 226; 497-509). For analysis of phospholipids, the lower organic phase was evaporated and the residue either subjected to phosphate determination (Chen, P.S., Toribara, T.Y. and Warner, H. (1956); Anal. Chem. 28, 1756-1758}, or dissolved in a small volume of chloroform-methanol for subsequent TLC
analysis of the phospholipids. Silica <~el plates (Merck) were used and were developed in the solvent system chloroform-methanol-acetic acid-water (25:15:4:2; v/v/v/v). The individual phospholipids were visualized by exposure to iodine and were identified on the basis of their RF values (comparison with reference samples). Protein was determined by a modified Lowry procedure (Peterson, G.L. (1977); Anal.
Biochem. 83, 346-356).
A total of 173 ml of solution (II) was mixed with~.the same volume of said fusion buffer solution (I). This influenza vi-rus dilution was pelletted by ultracentrifugation at 100,000 x g for 10 minutes.
15.3 ml of a detergent solution containing 54 mg/ml (=100 umol/mi) of octaethylene glycol monododecyl ether (OEG) in said fusion buffer solution was added to the influenza virus pellet. This corresponds to 18 p.g = 33.3 nmol OEG per ~g hemagglutinin. After 10 minutes, the pellet was completely dissolved. The solution was subjected to a 1 hour ultracen-. trifugation at 100,000 x g. 3000 mg of the dried antiviral drug dextran sulfate (Liischer, M. and Gliick, R., Antiviral Research 14, 39 - 50) were added to the remaining supernatant IV containing the solubilized influenza hemagglutinin trimer plus the non-essential neuraminidase residue and other con-stituents, making up solution V.
Phosphatidylcholine (SIGMA) was dissolved in a 2 . 1 mixture -of chloroform and methanol to a concentration of 10 mg/ml. 28 ml of this solution VI were carefully evaporated by applying a vacuum in a rotary evaporator, forming a thin phospholipid i0 layer of 280 mg on the inner surface of a round-bottom flask.
The solution V was now added to the phospholipid layer in the round-bottom flask. After shaking for at least 15 minutes and complete dissolution of the phosphatidylcholine, the resul-ting solution VII was transferred to a glass container toge-ther with 4.8 g of polystyrene beaded microcarriers PBM
having a mesh size (wet) of 25 - 50.
The container was then shaken in such a way that the content moved twice per second from one end to the other. One hour later, the suspension was aspirated with a thin pipette and transferred to a new container with 2.4 g of polystyrene bea-ded microcarriers of the same size. The column was shaken for minutes. This procedure was repeated twice. After the last 25 step, the resulting solution VIII was removed from the beads, fixed in a waterbath and treated with an ultrasonification apparatus (Bransonic, Branson Europe BV, frequency 50 kHz +10%). 10 seconds of ultrasonic shocks repeated twice, after intervals of 10 seconds each, yielded medium sized vesicles 30 with a diameter of about 50 - 100 nm. The final solution IX
was then diluted 1:100 with physiological NaCl solution X.

Example 2:
Preparation of synthetic membrane vesicles containing an antiviral antibody drug adsorbed with hemagglutinin trimers of influenza virus and CD4 monoclonal antibodies.
The vesicles were prepared according to Example 1 with the following modification: instead of 10 mg, only 9 mg of phos-phatidylcholine, and 1 mg of phosphati.dylethanolamine (kephaline) per ml were dissolved in a 2:1 mixture of chloroform and methanol.
After the preparation of hemagglutinin vesicles according to Example 1, 20 mg of sulfosuccinimidyl-~4-(N-maleimidophenyl)-butyrate (SMPB) (as a crosslinker) in 2 ml of water were added to the solution. After 1 hour at: room temperature under gentle shaking, the vesicles were pel7_etted by ult:racentrifu-gation during 10 min at 100,000 x g.
8 ml of Anti-Leu 3A (Becton & Dickinson) were added to the pellet. The resuspended material was carefully shaken for a few seconds every 5 minutes during one hour at room tempera-ture. Finally, the material was pe7:lei:,ted again (to remove non-bonded antibodies) by ultracentri:Eugation at .100,000 x g for 10 minutes. The pellet was resuspended in 1,500 ml of physiological NaCl solution.
Example 3:
Example 2 was repeated with the following modifications:
Instead of dextran sulfate, 1000 mg each of imidazol-carboxamide and hydroxy-urea (pharmaceuticals efficient against melanomas as described by Bru:nner and Nagel, Springer Verlag, 2nd edition, Internistische Kx~ebstherapie, page 93 (1979)) were added to the solution IV (see Example 1).

After adding the crosslinker and further processing as in Example 2, 1 mg of a monoclonal antibody (either R 24 as described by Houghton, A.N. et al. (198!5), Proc. Nat. Ac. Sc.
Vol. 82, p. 1242; or 0.5 mg L55 + 0.5 mg L?2 as decribed by Iric, R.S. et al., Lancet (1989), p. 786-787) were added to the vesicle solution VES to result in t7ze following total composition for 1000 human doses:
i0 46 mg hemagglutinin 250 mg phosphatidylcholine 30 mg phosphatidylethanolamine (kephaiine) 20 mg crosslinker (Sulfo - SMPB) 1 mg monoclonal antibody 1000 mg imidazol-carboxamide 1000 mg hydroxy-urea These pharmaceuticals so far conventionally had to be applied in about 5-fold quantitative dosage, i.e. the indicated quantities are for 200 human doses only.
Example 4:
Example 2 was repeated with the following modifications:
Instead of dextran sulfate, 1000 mg each of at least one of the following pharmaceuticals: adriplastin, endoxan, fluoro-uracil (as described by Brunner and Nagel, Internistische Krebstherapie; Springer-Verlag, 2nd edition, page 309, 7979) and colchicine (SIGMA) were added to the solution IV (see Example 1).
After adding the crosslinker and further processing as in Example 2, 1 mg of a monoclonal antibody JDB1 (as described by Strelkauskas, A.J., Cancer-Immunoi. Immunother. Vol. 23, p.31, 1986) was added to the vesicle solution VES to result in the following total composition for 1000 human doses of a ' pharmaceutical against mamma-carcinomas.
46 mg hemagglutinin 250 mg phosphatidylcholine 30 mg phosphatidylethanolamine 20 mg crosslinker (Sulfo - SMPB) 1 mg monoclonal antibody 1000 mg adriplastin 1000 mg endoxan 1000 mg fluoro-uracil Example 5:
Examples 1 and 2 were repeated with the following modific-ations:
Instead of influenza hemagglutinin trimers, the monomers in-cluding the fusion peptide were used. The hemagglutinin-2-monomer containing the fusion peptide was obtained by cleav-age of the S-H bridges by chemical reduction with D4-dithio-threitol (DTT, Sigma) and subsequent separation from the hem-agglutinin-1 peptide by gel chromatography (Sephadex G 50) at pH 6. The purified hemagglutinin-2-monomer suspension con-tained 180 ug of monomers including the fusion peptide.
Example 6:
Example 2 was repeated with the following modifications:
Instead of influenza hemagglutinin trimers, the crude fusion peptide was used. The influenza fusion peptide used for pre-paration of synthetic membrane vesicles was obtained by che-mical synthesis. Any one of the aminoacid sequences listed in FiQ.2 may be used. The arrangement of at least one, prefer-ably three, cystein groups at one end of the respective se-quence has been found useful for the fusion activity.
The solution with one of these synthetic fusion peptides con-s tained 4 ug per ml.
The vesicles containing one of the abo«e fusion peptides have been prepared as follows:
9 mg of phosphatidyicholine and 1 mg oi= phosphatidylethanol-amine per ml were dissolved in a 2:1 m_Lxture of chloroform and methanol. 28 ml of this solutibn wESre carefully evapor-ated by applying a vacuum in a rotary evaporator forming a thin phospholipid layer on the inner surface of a round-bottom flask.
3 g of dextran sulfate were dissolved :in 15.3 ml of a deter-gent solution containing 100 umoi of octaethylene glycol monododecyl ether per 1 ml of fusion buffer; the solution was then added to the phospholipid layer in the round-bottom flask. After shaking for at least 15 minutes and complete dissolution of the phospholipid layer, the solution was transferred to a glass container toget'.her with 4.8 g of polystyrene beaded microcarriers having a mesh size (wet) of 25-50.
The container was then shaken in such a way that the content moved twice per second from one end to the other. One hour later, the suspension was~aspirated with a thin pipette and 30. transferred to a new container with 2.4 g of polystyrene bea-ded microcarriers of the same size. The container was shaken for 30 minutes. This procedure was repeated twice. After the last step, the resulting solution was removed from the beads, fixed in a waterbath and treated with an ultrasonification apparatus (Bransonic, Branson Europe BV, frequency 50 kHz +10~). 10 seconds of ultrasonic shocks repeated twice, after _ 17 _ intervals of 10 seconds each, yielded medium size vesicles with a diameter of about 50-100 nm.
2 ml of water containing 20 mg of sulfa-SMPB tpierce) were added to the above suspension. After 1 hour at room temperature under gentle shaking, the vesicles were pelletted by 15 minutes of ultracentrifugation at: 100,000 x g.
2 ml of the solution containing the synthetic fusion peptide were added to the pellet. The resuspen~ied material was care-fully shaken for a few seconds every 5 minutes during 1 hour at room temperature. Finally, the material was pelletted again (to remove non-bonded fusion peptides) by ultracentrifugation at 100,000 x g for 10 minutes. The pellet was resuspended in 200 ml of physiological NaCl solution.
Example 7:
Example 6 was repeated with the following modifications:
2 ml of a solution containing 2 ug of a synthetic fusion pep-tide and 2 ml containing 1OO ~g of Anti-Leu 3A were added to the pellet containing the vesicles. The resuspended material was carefully shaken for a few seconds every 5 minutes during one hour at room temperature. Finally, the material was pelletted again (to remove non-bonded :fusion and cell specific peptides) by ultracentrifugation at 100,000 x g for 10 minutes. The pellet was resuspended in 200 ml of physiological NaCl solution.
Example 8:
Vesicles according to Examples 1 through 4 were prepared with the fusion peptide or hemagglutinin from rhabdoviruses, parainfluenzaviruses or togaviruses.

WO 92/13525 ' PCT/EP92/00089 _ 18 _ a) The rhabdovirus rabies was producet~ in human diploid cells. The harvests (supernatants) containing 107 rabies viruses per ml were purified and concentrated by sucrose density ultracentrifugation. The purified rabies virus suspension contained 210 ~.tg of rabies virus hemagglutinin per ml and was further processed according t:o Example 1.
b) The parainfluenza virus type III was grown in the allan-toic cavity of hen eggs and was purified twice by ultra-centrifugation in a sucrose gradient as in Example 1. The purified parainfluenza virion suspension contained 245 y~g of parainfluenza virus hemagglutinin per ml and was further processed according to Example 1.
c) The togavirus rubella was produced in human diploid cells and purified according a). The purified rubella virion suspension contained 205 ug of rubella 'virus hemagglutinin per ml and was further processed according to Example 1.
Example 9:
Antiviral drugs were prepared according to Example 1. hu-PBL-SCID mice were treated for 14 days with ribonuclease dimer for 10 days alternatively with a phosphate buffer solution (blank test) only, with dextran sulfate or ribonuclease dimer (prepared according to Example 1 of PCT/US90/00141) at three dose levels, with dextran sulfate in liposomes, or ribonuclease dimer in liposomes, both latter prepared ac-cording to Example 1 of the present invention. Treatment was initiated at the same time as virus challenge with 100 TCID50 of HIV-1IIIB. hu-PBL-SCID mice were assessed for evidence of viral infection (virus isolation, PCR detection of proviral sequences) at 2, 4, and 6 weeks following viral infection.
The results of the study show the percentage of animals in each treatment group from which virus was isolated:

w0 92/13525 PCT/EP92/00089 phosphate buffer solution 80$
(blank test) ribonuclease dimer 0.001 mg/kg 92g ribonuclease dimer 0.1 mg/kg 30~

ribonuclease dimer 10.0 mg/kg 63~

dextran sulfate 10 mg/kg 33%

dextran sulfate 10 mg/kg in liposomes 14~

ribonuclease dimer in liposomes 25~

The study results indicate protection of the majority of ~0 treated hu-PBL-SCID mice from HIV infection following q. 12 hr. injection of 0.1 mg/kg ribonuclease dimer, l0 mg/kg dex-tran sulfate, liposomes containing ribonuclease dimer, and liposomes containing dextran sulfate. Protection was marginal with ribonuclease dimer at 10 mg/kg, which might be due to the fact that, at higher dosage levels, ribonuclease dimer suppresses immunity. No protective effect of ribonuclease di-mer at 0.001 mg/kg was seen. However, with ribonuclease dimer in liposomes according to the present invention, the success rate improved from 63% to 25% in spite of the high dosage.
Further improvement is expected from optimum dosage levels in liposomes.
The same conclusions hold when mice with poorly functioning human PBL grafts at the end of the experiment are excluded from analysis, although it appears from analysis of human im-munoglobulin levels and (3-globin PCR results that treatment with either ribonuclease dimer liposomes or dextran sulfate liposomes interfered with the survival. of human cells. Exclu-sion of hu-PBL-SCID mice on the basis of human function al-lows some distinction between direct anti-viral effects and immunomodulatory activity.
Example 10:
In a fusion test described by Luscher and Gluck (1990) (Anti-virai Research 14, 39 - 50), vesicles prepared according to ..O 92/13525 PCT/EP92/00089 Example 1 were compared with reconstituted influenza vesicles prepared according to the method of Kawasaki et al. in fusion activity with model membranes:
kinetics of fluorescence de- uenching of 818-Fig..3 shows the q labelled influenza A virus with DOPC-cholesterol liposomes.
The increase in fluorescence is expressed in $ FDQ, cal-culated according to Luscher & Gluck (see above).
The initial fusion rates were obtained from the tangents to the fusion curves at time 0, when the fusion was initiated (dotted line in Fig.3). Curve 2 corresponds to the fusion ac-tivity of vesicles prepared according to the method of Ka-wasaki et al.

Claims (42)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. A phospholipid bi-layer vesicle having at least one desired drug or pharmaceutically-active substance therein, the vesicle comprising:
(a) a membrane having one or more viral phospholipids in combination with other phospholipids comprising phosphatidylethanolamine (PE);
(b) at least one active fusion peptide that is a non-Sendai viral hemagglutinin that causes the vesicles to be internalized by target cells through phagocytosis or endocytosis, said hemagglutinin being attached directly or indirectly to said membrane;
(c) a bifunctional crosslinker linked to phosphatidylethanolamine (PE) of said membrane; and (d) at least one cell-specific marker linked via its sulphur to the PE-bound crosslinker, said marker being a biologically active protein for binding to a receptor of target cells.
2. The vesicle according to claim 1, whose membrane comprises less than 1% cholesterol by weight.
3. The vesicle according to claim 1 or 2, wherein said vesicle has a diameter ranging from about 50 to 100 nm.
4. The vesicle according to claim 1, 2 or 3, wherein the viral phospholipids are derived from at least one virus selected from the group consisting of influenza virus, rhabdovirus, parainfluenza virus type III, Semliki Forest virus and togavirus.
5. The vesicle according to claim 4, wherein the influenza virus is of the A-H1 N1 subtype.
6. The vesicle according to any one of claims 1 to 5, wherein the membrane includes:
70-95% by weight of phosphatidylcholine and 5 to 30% by weight of phosphatidylethanolamine, based on total phospholipids; and to 10% by weight of said bifunctional crosslinker.
7. The vesicle according to any one of claims 1 to 6, wherein the crosslinker is a sulfosuccinimidyl derivative.
8. The vesicle according to any one of claims 1 to 7, wherein the cell-specific marker is a monoclonal antibody.
9. The vesicle according to claim 8, wherein said antibody is an IgG antibody.
10. The vesicle according to any one of claims 1 to 9, wherein said other phospholipids further comprise phosphatidylcholine.
11. The vesicle according to any one of claims 1 to 10, wherein the desired drug or pharmaceutically-active substance is selected from the group consisting of dextran sulfate, ribonuclease dimer, lysozyme dimer, imidazole-carboxamide, hydroxy-urea, adriblastin, endoxan, fluoro-uracil, and colchicine.
12. The vesicle according to any one of claims 1 to 11, wherein said viral hemagglutinin is hemagglutinin derived from at least one virus selected from the group consisting of influenza virus, rhabdovirus, parainfluenza virus type III, Semliki Forest virus, and togavirus.
13. The vesicle according to any one of claims 1 to 12, wherein said viral hemagglutinin is hemagglutinin trimer of influenza virus.
14. The vesicle according to any one of claims 1 to 13, wherein the bifunctional crosslinker is derived from a crosslinking organic molecule that comprises a carboxylic and a maleimido group.
15. The vesicle according to claim 14, wherein the organic molecule is sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (Sulfo-SMPB).
16. The vesicle according to any one of claims 1 to 15, wherein said other phospholipids further comprise phosphatidylcholine which is present in the membrane in a weight-ratio ranging from 1:2 to 1:100 of viral phospholipids:phosphatidylcholine.
17. A pharmaceutical composition, comprising a pharmaceutically-acceptable carrier and an effective amount of a desired drug or pharmaceutically-active substance encapsulated in a vesicle as defined in any one of claims 1 to 16, wherein the effective amount ranges from 0.001 to mg of drug per kg body weight.
18. The pharmaceutical composition according to claim 17, wherein the desired drug is an anti-cancer drug.
19. The pharmaceutical composition according to claim 17, wherein the desired drug is an anti-viral drug.
20. A process for the preparation of a phospholipid bi-layer vesicle comprising at least one fusion peptide and at least one cell-specific marker on the membrane, and at least one desired drug or pharmaceutically-active substance, the process comprising the steps of:
(a) dissolving purified virus, or parts thereof, containing non-Sendai hemagglutinin that causes the vesicles to be internalized by target cells by phagocytosis or endocytosis, in a non-ionic detergent solution that does not react with hemagglutinin and that comprises octaethyleneglycol monododecylether;
(b) subjecting the solution resulting from step (a) to ultracentrifugation, and mixing the resulting supernatant which contains viral lipids and at least one hemagglutinin fusion peptide with said desired drug or substance;
(c) combining the mixture with other phospholipids, said other phospholipids comprising phosphatidylethanolamine;
(d) repeatedly treating the mixture from step (c) with microcarriers to remove the detergent whereby vesicles are formed;
(e) subjecting the vesicles resulting from step (d) to repeated ultrasonication to adjust the size of the vesicles;
(f) reacting the vesicles of step (c) with a bifunctional crosslinker for binding to phosphatidylethanolamine (PE) of the vesicle membrane and for binding polypeptides, and pelleting the vesicles; and (g) reacting the pelleted vesicles with a solution containing at least one cell-specific marker for binding to the PE-bound crosslinker, said marker being a biologically active protein for binding to a receptor of target cells.
21. The process according to claim 20, wherein the detergent solution comprises a fusion buffer and 10 to 250 µmol octaethyleneglycol monododecylether per ml.
22. The process according to claim 21, wherein the detergent solution comprises a fusion buffer and 80 to 120 µmol octaethyleneglycol monododecylether per ml.
23. The process according to claim 20, 21 or 22, wherein the microcarriers are polystyrene beaded microcarriers having a wet mesh size of 20-50, and the solution under step (d) is treated four times with said microcarriers.
24. The process according to any one of claims 20 to 23, wherein said cell-specific marker is an antibody.
25. The process according to any one of claims 20 to 24, wherein the crosslinker is a sulfosuccinimidyl derivative.
26. The process according to any one of claims 20 to 25, wherein the detergent.is removed from the solution by the application of 1 to 2 g of polystyrene beaded microcarriers per 100 mg detergent.
27. The process according to any one of claims 20 to 26, wherein said other phospholipids added in step (c) further comprise phosphatidylcholine.
28. The process according to any one of claims 20 to 27, wherein said viral hemagglutinin is hemagglutinin derived from at least one virus selected from the group consisting of influenza virus, rhabdovirus, parainfluenza virus, Semliki Forest virus and togavirus.
29. The process according to claim 28, wherein said viral hemagglutinin is hemagglutinin trimer of influenza virus.
30. The process according to any one of claims 20 to 29, wherein the crosslinker is a crosslinking organic molecule that comprises a carboxylic and a maleimido group.
31. The process according to claim 30, wherein said organic molecule is a sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (Sulfo-SMPB).
32. A phospholipid bi-layer vesicle comprising:
at least one fusion peptide and at least one cell-specific marker on a membrane; and at least one desired drug or pharmaceutically-active substance, wherein the membrane includes:
(a) one or more viral phospholipids in combination with other phospholipids, the other phospholipids comprising phosphatidylethanolamine;
(b) at least one non-Sendai viral hemagglutinin, as a fusion peptide;
(c) a bifunctional crosslinker bound to said membrane containing said phosphatidylethanolamine, said crosslinker for binding polypeptides;
(d) at least one cell-specific marker linked via sulphur to the crosslinker, said marker being a protein which is further capable of linking to a receptor of cells inducing the endocytosis mechanism;
and (e) cholesterol in a concentration of less than approximately loo by weight.
33. The vesicle according to claim 32, wherein said other phospholipids further comprise phosphatidylcholine which is present in the membrane in a weight-ratio ranging from 1:2 to 1:100 of viral phospholipids:phosphatidylcholine.
34. A phospholipid bi-layer vesicle of a structure that results from the steps of:

(a) dissolving purified virus or parts thereof containing non-Sendai hemagglutinin that causes the vesicles to be internalized by target cells by phagocytosis or endocytosis, in a non-ionic detergent solution that does not react with hemagglutinin and that comprises octaethyleneglycol monododecylether;
(b) subjecting the solution resulting from step (a) to ultracentrifugation, and mixing the resulting supernatant which contains viral lipids and at least one hemagglutinin fusion peptide with said desired drug or substance;
(c) combining the mixture with other phospholipids, said other phospholipids comprising phosphatidylethanolamine;
(d) repeatedly treating the mixture from step (c) with microcarriers to remove the detergent whereby vesicles are formed;
(e) subjecting the vesicles resulting from step (d) to repeated ultrasonication to adjust the size of the vesicles;
(f) reacting the vesicles of step (e) with a bifunctional crosslinker for binding to phosphatidylethanolamine (PE) of the vesicle membrane and for binding polypeptides, and pelleting the vesicles; and (g) reacting the pelleted vesicles with a solution containing at least one cell-specific marker for binding to the PE-bound crosslinker, said marker being a biologically active protein for binding to a receptor of target cells.
35. Use of phospholipid bi-layer vesicles as defined in any one of claims 1 to 16 and 32 to 34, for slowing the progression of cancer, wherein at least one anti-cancer drug is encapsulated in said vesicles and is formulated for administration to a patient at a dose of approximately 0.001 to 10 mg of drug per kg body weight.
36. The use according to claim 35, wherein said anti-cancer drug is selected from the group consisting of dextran sulfate, ribonuclease dimer, lysozyme dimer, imidazole-carboxamide, hydroxy-urea, adriblastin, endoxan, fluoro-uracil, and colchicine.
37. Use of phospholipid bi-layer vesicles as defined in any one of claims 1 to 16 and 32 to 34, for prophylactic intervention or therapeutic treatment of a viral disease, wherein at least one anti-viral drug is encapsulated in said vesicles and is formulated for administration to a patient at a dose of approximately 0.001 to 10 mg of drug per kg body weight.
38. The use according to claim 37, wherein said anti-viral drug is selected from the group consisting of dextran sulfate, ribonuclease diner and lysozyme diner.
39. Use of phospholipid bi-layer vesicles as defined in any one of claims 1 to 16 and 32 to 34, in the manufacture of a medicament for slowing the progression of cancer, wherein at least one anti-cancer drug is encapsulated in said vesicles and is formulated for administration to a patient at a dose of approximately 0.001 to 10 mg of drug per kg body weight.
40. The method according to claim 39, wherein said anti-cancer drug is selected from the group consisting of dextran sulfate, ribonuclease dimer, lysozyme dimer, imidazole-carboxamide, hydroxy-urea, adriblastin, endoxan, fluoro-uracil, and colchicine.
41. Use of phospholipid bi-layer vesicles as defined in any one of claims 1 to 16 and 32 to 34, in the manufacture of a medicament for prophylactic intervention or therapeutic treatment of a viral disease, wherein at least one anti-viral drug is encapsulated in said vesicles and is formulated for administration to a patient at a dose of approximately 0.001 to 10 mg of drug per kg body weight.
42. The use according to claim 41, wherein said anti-viral drug is selected from the group consisting of dextran sulfate, ribonuclease dimer and lysozyme dimer:
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